1 Field of the Invention
This invention relates to the use of recombinant DNA technology to produce yeast strains that will produce wheat gluten proteins and to plasmids which include genes coding for wheat gluten proteins.
2. Description of the Art
Recently developed recombinant deoxyribonucleic acid (DNA) techniques have made it possible to utilize microorganisms for synthesis of commercially useful proteins and peptides which are normally made by other organisms. Briefly, the procedure involves: (1) isolation and purification of the specific gene or gene segment containing the genetically coded information for the amino acid sequence of the desired protein, (2) recombination of the isolated gene segment with an appropriate transfer vector, and (3) transfer of the vector to the appropriate microorganism and selection of a strain of microorganism containing the desired information.
Examples of foreign genes expresssed in yeast include the wheat .alpha.-amylase gene (Rothstein et al., Nature 308: 662-665 (1984)), human epidermal growth factor (Brake et al., Proc. Natl. Acad. Sci. U.S.A. 81: 4642-4646 (1984)), chicken egg white lysozyme (Oberto and Davidson, Gene 40: 57-65 (1985)), and Torpedo californica acetylcholine receptor .alpha. subunit (Fujita et al., Science 231: 1284-1287 (1986)). A fragment of the maize seed storage protein, zein, has also been produced in yeast (Coraggio et al., EMBO J. 5: 459-465 (1986)).
There is no report in the prior art of expression of a wheat gluten gene in lower eukaryotes, for example yeast. Wheat gluten proteins consist of gliadin and glutenin proteins. The gliadins are monomeric proteins of 30,000 to 78,000 molecular weight; they comprise a multigene family and have been historically assigned to .alpha., .beta., .gamma., and w classes based on electrophoretic mobility. They constitute a major fraction of the seed storage proteins of hexaploid wheat grain and are important dietary proteins and determinants of dough and bread baking quality. In addition, gliadins, or peptides derived from them during digestion, initiate damage to the absorptive epithelium of the small intestine to produce symptoms of celiac disease in susceptible individuals.
While production of wheat gluten proteins by yeast strains would be of considerable value for production of pure proteins for use in diagnosis and treatment of celiac disease, for testing theories of dough formation, or for supplementation of baking formulations, heretofore no yeast having this capability has been made. Problems inherent in the expression of a wheat gluten gene in yeast include the following: (1) extreme differences in amino acid codon preference between gluten protein mRNAs and abundant yeast protein mRNAs make predictions regarding the compatibility of gluten mRNAs with the yeast protein synthesis system impossible; (2) lack of prior knowledge regarding the stability of wheat gluten protein mRNAs in yeast make predictions of the actual synthesis of complete gluten protein molecules in yeast impossible; (3) lack of prior knowledge of the toxicity, if any, of wheat gluten proteins to yeast make predictions of the maintenance of viable wheat gluten protein-producing yeast strains impossible; and (4) lack of knowledge of the compatibility of the yeast .alpha.-gliadin precursor with the protein processing systems of yeast make it impossible to predict whether the signal peptide would be cleaved from the .alpha.-gliadin precursor to produce the mature protein.